1. Field of the Invention
The present invention relates to a method for analyzing L-threonine, and also to L-threonine dehydrogenase that can be employed in this analysis method.
2. Brief Description of the Related Art
L-threonine, an essential amino acid, must be obtained from food. L-threonine is necessary to maintain the balance of nitrogen within the body and to promote healthy growth. It also performs functions in the cardiovascular system, liver, central nervous system, intestines, and immune system.
Vitamin B12 deficiency, type II citrullinemia, sepsis, and amino acid or nitrogen imbalances cause deficiencies in the threonine content of the blood. Further, when vegetarians eat grains with low quantities of threonine-containing materials, they sometimes develop threonine deficiencies. The quantification of L-threonine is required for the diagnosis of various illnesses and congenital metabolic disorders, providing long-term dietary supplements to patients, research relating to illnesses involving amino acid metabolic disorders, and the like.
Various methods of quantifying L-threonine have been reported. Protein hydrolysate, gelatin, and blood threonine can be quantified by the conversion of threonine to acetaldehyde by lead tetraacetate, absorption by concentrated sulfuric acid, the measurement of pigment generated by the condensation of p-hydroxybiphenyl and acetaldehyde, high performance liquid chromatography, mass spectrometry, amino acid analyzer, and the like. These methods present problems in that they are dangerous to operate, require numerous steps, utilize expensive equipment, and are not suited to mass screening involving the handling of large numbers of samples.
There is an enzymatic method that employs threonine deaminase (EC 4.2.1.16). This enzyme degrades L-threonine into α-ketobutyrate and ammonia. Thus, a method that converts the α-ketobutyrate that is produced into hydrazone derivatives has been reported (Watanabe K, Itoh N, Tanaka A, Fukui S. Application of an immobilized Escherichia coli cell tube in analysis of L-threonine. Agric. Biol. Chem. (1982) 46:119-126.).
There is an example in which threonine in rat plasma is oxidized by periodic acid, and the aldehyde produced is quantified by aldehyde dehydrogenase (EC 1.2.1.5). The remainder of the periodic acid is consumed by the addition of D-galactose (Nishida T, Kume S, Saito M, Suda M. A specific method for the determination of threonine in rat blood plasma using aldehyde dehydrogenase. J. Biochem. (1977) 81:1085-1090). Acetaldehyde produces NADH by the reduction of NAD+ through the action of aldehyde dehydrogenase, and is quantified by a method employing a fluorescent pigment.
However, in the method described in Watanabe et al. (Application of an immobilized Escherichia coli cell tube in analysis of L-threonine. Agric. Biol. Chem. (1982) 46:119-126), threonine deaminase has activity not just on L-threonine, but also on L-serine and D-serine. Thus, this method is unsuited to the quantification of samples containing L-serine and the like.
In the method described in Nishida et al. (A specific method for the determination of threonine in rat blood plasma using aldehyde dehydrogenase. J. Biochem. (1977) 81:1085-1090), the remaining periodic acid must be consumed by adding D-galactose. It is thus not a quantification method based on a single enzyme.